High nonpublication rate from publication professionals hinders evidence-based publication practices.

Background. The need for timely, ethical, and high-quality reporting of clinical trial results has seen a rise in demand for publication professionals. These publication experts, who are not ghostwriters, work with leading medical researchers and funders around the world to plan and prepare thousands of publications each year. Despite the involvement of publication professionals in an increasing number of peer-reviewed publications, especially those that affect patient care, there is limited evidence-based guidance in the peer-reviewed literature on their publication practices. Similar to the push for editors and the peer-review community to conduct and publish research on publication ethics and the peer-review process, the International Society for Medical Publication Professionals (ISMPP) has encouraged members to conduct and publish research on publication planning and practices. Our primary objective was to investigate the publication rate of research presented at ISMPP Annual Meetings. Methods. ISMPP Annual Meeting abstract lists (April 2009-April 2014) were searched in November 2014 and data were extracted into a pilot-tested spreadsheet. MEDLINE was searched in December 2014 to determine the publication rate (calculated as the % of presented abstracts published as full papers in peer-reviewed journals). Data were analyzed using the Cochran-Armitage trend test (significance: P < .05) by an independent academic statistician. Results. From 2009 to 2014, there were 220 abstracts submitted, 185 accepted, and 164 presented. There were four corresponding publications (publication rate 2.4%). Over time, ISMPP's abstract acceptance rate (overall: 84.1%) did not change, but the number of abstracts presented increased significantly (P = .02). Most abstracts were presented as posters (81.1%) and most research was observational (72.6%). Most researchers came from the US (78.0%), followed by Europe (17.7%), and the Asia-Pacific region (11.2%). Discussion. Research presented at ISMPP Annual Meetings has rarely been published in peer-reviewed journals. The high rate of nonpublication by publication professionals has now been quantified and is of concern. Publication professionals should do more to contribute to evidence-based publication practices, including, and especially, their own. Unless the barriers to publication are identified and addressed, the practices of publication professionals, which affect thousands of peer-reviewed publications each year, will remain hidden and unproven.

Sponsor-Imposed Publication Restrictions Disclosed on ClinicalTrials.gov.

We investigated whether sponsor-imposed publication restrictions for ClinicalTrials.gov trials were reasonable, based on consistency with Good Publication Practice 2 (GPP2). ClinicalTrials.gov trial record data were electronically imported (October 7, 2012) and screened for eligibility (phase 2-4, interventional, recruitment closed, results available, first received for registration after November 10, 2009, any sponsor type, investigators not sponsor employees). Two authors categorized restrictions information as consistent or not consistent with GPP2, resolving discrepancies by consensus. Of the eligible trials (388/484, n = 81,768 participants), 80.7% (313/388) had restrictions disclosed, and 92.5% (311/388) were industry-sponsored. Significantly more trials had restrictions that were consistent with GPP2 than not (74.1% [232/313], n = 55,280 participants vs. 25.9% [81/313], n = 19,677 participants; P < .001). Reasons for inconsistency were insufficient, unclear, or ambiguous information (48.1%, 39/81), sponsor-required approval for publication (35.8%, 29/81), sponsor-required text changes (8.6%, 7/81), and outright bans (7.4%, 6/81). Follow-up of trials with insufficient information and a contact email (response rate, 46.9% [15/32]) revealed 2 additional bans. A total of 776 participants had consented to trials that had publication bans. Many, but not all, sponsor-imposed publication restrictions disclosed on ClinicalTrials.gov may be considered reasonable. Sponsors should ensure restrictions are appropriately disclosed. Volunteers should be alerted to any restrictions before consenting to participate in a clinical trial.

Antenatal glucocorticoid exposure enhances the inhibition of adrenal steroidogenesis by leptin in a sex-specific fashion.

Antenatal treatment with glucocorticoids (GC) poses long-lasting effects on endocrine and cardiovascular function. Given that leptin attenuates adrenal function and the reported sex differences in plasma leptin concentration, we hypothesized that antenatal GC will affect leptin levels and leptin modulation of adrenal function in a sex-specific manner. Pregnant sheep were randomly given betamethasone or vehicle at 80 days of gestational age, and offspring were allowed to deliver at term. Adrenocortical cells (ADC) were studied from male and female animals at 1.5 yr of age. Plasma leptin was increased 66% in male and 41% in female GC-treated animals (P < 0.05), but adrenal leptin mRNA was increased only in GC-treated males (P < 0.05). Whereas mRNA expression of adrenal leptin receptor isoforms showed sex (Ob-Ra and Ob-Rb) and treatment-dependent (Ob-Rb) differences, protein expression remained unchanged. GC-treated females showed greater plasma cortisol and greater ACTH-stimulated cortisol production (P < 0.05) in ADC. Leptin exerted a greater inhibitory effect on basal and stimulated cortisol by ADC from GC-treated males (P < 0.05), with no differences in females. Similarly, greater inhibitory effects on basal and ACTH-stimulated StAR and ACTH-R mRNA expression by leptin were observed in cells from GC males (P < 0.05), with no changes in females. Persistent effects of antenatal GC on leptin levels and leptin modulation of adrenal function are expressed in a sex-specific manner; males are more sensitive than females to the inhibitory influences of leptin on adrenal function, and this effect appears to be mediated by a greater inhibition of StAR and ACTH-R expression in adrenals of adult GC-treated males.

Antenatal betamethasone exposure alters renal responses to angiotensin-(1-7) in uninephrectomized adult male sheep.

Antenatal corticosteroid exposure reduces renal function and alters the intrarenal renin-angiotensin system to favor angiotensin activation of angiotensin type 1 receptor (AT1R) mediated responses in ovine offspring. This study aimed to assess whether antenatal steroid exposure would affect renal responses to the direct intrarenal infusion of angiotensin-(1-7) in rams and the angiotensin receptors involved in mediating responses to the peptide. Adult, uninephrectomized rams exposed to either betamethasone or vehicle before birth received intrarenal angiotensin-(1-7) infusions (1 ng/kg/min) alone or in combination with antagonists to angiotensin receptors for 3 h. Basal sodium excretion (UNa) was significantly lower and mean arterial pressure was significantly higher in betamethasone- compared to the vehicle-treated sheep. Angiotensin-(1-7) decreased UNa more in betamethasone- than in vehicle-treated sheep. Candesartan reversed the response to angiotensin-(1-7) but D-Ala(7)-angiotensin-(1-7) did not. Angiotensin-(1-7) infusion decreased effective renal plasma flow in both groups to a similar extent and the response was reversed by candesartan, but was not blocked by D-Ala(7)-angiotensin-(1-7). Glomerular filtration rate increased significantly in both groups after 3 h infusion of angiotensin-(1-7) plus candesartan. These results suggest that antenatal exposure to a clinically relevant dose of betamethasone impairs renal function in rams. Moreover, angiotensin-(1-7) appears capable of activating the AT1R in uninephrectomized rams.

Leptin alters adrenal responsiveness by decreasing expression of ACTH-R, StAR, and P450c21 in hypoxemic fetal sheep.

The late gestation increase in adrenal responsiveness to adrenocorticotropin (ACTH) is dependent upon the upregulation of the ACTH receptor (ACTH-R), steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes in the fetal adrenal. Long-term hypoxia decreases the expression of these and adrenal responsiveness to ACTH in vivo. Leptin, an adipocyte-derived hormone which attenuates the peripartum increase in fetal plasma cortisol is elevated in hypoxic fetuses. Therefore, we hypothesized that increases in plasma leptin will inhibit the expression of the ACTH-R, StAR, and steroidogenic enzymes and attenuate adrenal responsiveness in hypoxic fetuses. Spontaneously hypoxemic fetal sheep (132 days of gestation, PO(2) ≈ 15 mm Hg) were infused with recombinant human leptin (n = 8) or saline (n = 7) for 96 hours. An ACTH challenge was performed at 72 hours of infusion to assess adrenal responsiveness. Plasma cortisol and ACTH were measured daily and adrenals were collected after 96 hours infusion for messenger RNA (mRNA) and protein expression measurement. Plasma cortisol concentrations were lower in leptin- compared with saline-infused fetuses (14.8 ± 3.2 vs 42.3 ± 9.6 ng/mL, P < .05), as was the cortisol:ACTH ratio (0.9 ± 0.074 vs 46 ± 1.49, P < .05). Increases in cortisol concentrations were blunted in the leptin-treated group after ACTH(1-24) challenge (F = 12.2, P < .0001). Adrenal ACTH-R, StAR, and P450c21 expression levels were reduced in leptin-treated fetuses (P < .05), whereas the expression of Ob-Ra and Ob-Rb leptin receptor isoforms remained unchanged. Our results indicate that leptin blunts adrenal responsiveness in the late gestation hypoxemic fetus, and this effect appears mediated by decreased adrenal ACTH-R, StAR, and P450c21 expression.

The midgestational maternal blood pressure decline is absent in mice lacking expression of the angiotensin II AT2 receptor.

The midgestational maternal blood pressure (BP) decrease is absent in mice treated with an angiotensin II AT2 receptor blocker. We tested the hypotheses that there would be 1) no midgestational decrease in maternal systolic BP (SBP) in AT2-/- mice, and 2) a pattern of increased AT2 and/or decreased AT1a mRNA expression in tissues from normal (wild-type, WT) mice, corresponding with SBP changes. Heart, aorta, placenta and kidney tissue were obtained from WT and AT2-/- mice before pregnancy and on gestational days (Gd) 5-6, 12-13 and 18-19. AT1a and AT2 mRNA expression was quantified. SBP was measured. SBP was significantly decreased in WT Gd12-13 mice, but did not change during pregnancy in AT2-/- mice. In WT mice, aortic AT1a mRNA expression levels were significantly higher at Gd12-13 and Gd18-19 compared with before pregnancy. AT1a kidney and heart mRNA did not change during pregnancy. There were no changes in AT2 mRNA expression. There was no distinct pattern of change in AT1a expression in AT2-/mice. Placental AT1a and AT2 expression levels increased markedly between Gd12-13 and Gd18-19 in WT mice. We conclude that the AT2 receptor is essential for the midgestational SBP decline in WT mice. There is no consistent relationship between changes in tissue angiotensin II receptor mRNA expression and SBP in WT mice.

Fetal and postnatal renin secretion in female sheep exposed to prenatal betamethasone.

Prenatal glucocorticoids have long-term effects on the kidney and blood pressure that may be mediated by the renin-angiotensin system (RAS). We studied the effects of antenatal betamethasone administration on renin in fetal and adult female sheep. Pregnant sheep received 2 doses of betamethasone or vehicle, at 80 and 81 days of gestation (dGA). Fetuses were delivered within 24 hours following treatment, at 135 dGA, or allowed to continue to term. Plasma and kidney samples were collected from fetal and 1-year-old sheep. Plasma and renal renin and renin messenger RNA (mRNA) were measured. Significant decreases in plasma and renal renin and renin mRNA were apparent in female betamethasone fetuses at 80 dGA (P < .05). At 135 dGA, renal renin concentrations were significantly increased in betamethasone fetuses. At 1 year, renin levels were similar in the 2 groups. These findings suggest that prenatal betamethasone has an immediate effect on expression and secretion of renin. The downregulation of renin at 80 dGA may affect nephron development.

Ethanol-mediated fetal dysmorphology and its relationship to the ontogeny of maternal liver metallothionein.

BACKGROUND: Fetal zinc (Zn) deficiency arising from ethanol-induction of the Zn-binding protein metallothionein (MT) in the mother's liver has been proposed as a mechanism of teratogenicity. Here, we determine the ontogeny of MT and Zn homeostasis in rats and mice and then examine the effect of acute ethanol exposure in early embryonic development on this relationship. The protective effect of Zn against ethanol-mediated fetal dysmorphology is also examined. METHODS: Study 1: Maternal liver MT and Zn homeostasis was determined in Sprague-Dawley rats and C57BL/6J mice throughout gestation. Study 2: Rats were administered ethanol (25% in saline, intraperitoneal 0.015 ml/g) or vehicle alone on gestational day (GD) 9. Maternal liver MT and Zn, and plasma Zn was determined over the ensuing 24 hours. Study 3: Pregnant rats were treated with ethanol and Zn (s.c. 2.5 microg Zn/g) on GD9 and fetal dysmorphology was assessed on GD 19. RESULTS: Study 1: Maternal liver MT began to rise around GD 9 peaking on GD 15 before falling to nonpregnant levels around term. The pregnancy-related increase in MT was associated with a fall in plasma Zn which was significantly lower on GD 15 thereafter returning to nonpregnant levels by parturition. Study 2: Ethanol administered to pregnant rats on GD 9 resulted in a 10-fold induction of MT in the maternal liver and was associated with a 33% rise in liver Zn and a 30% fall in plasma Zn, 16 hours after treatment. Study 3: Ethanol treatment on GD 9 resulted in a significant increase in craniofacial malformations which were prevented by concurrent Zn treatment. CONCLUSIONS: The findings indicate that maternal liver MT levels are lowest in early gestation (before GD 10) making this a sensitive period where ethanol-induction of MT can affect fetal Zn homeostasis and cause fetal dysmorphology. The study further provides evidence of a protective role for Zn against ethanol-mediated teratogenicity.

Gender differences in the effects of antenatal betamethasone exposure on renal function in adult sheep.

Exposure to clinically relevant doses of glucocorticoids during fetal life increases blood pressure in adult male and female sheep. The purpose of this study was to evaluate the effects of prenatal exposure to betamethasone at 80-81 days of gestation on renal function in ewes and rams at 1.5 yr of age. In prenatal betamethasone-exposed males, compared with the vehicle-exposed animals, basal glomerular filtration rate (GFR) (1.93 +/- 0.08 vs. 2.27 +/- 0.10 ml.min(-1).kg body wt(-1)) and the ability to excrete an acute Na+ load (37.1 +/- 4.4 vs. 53.7 +/- 9.7%) were reduced. (P < 0.03 and P = 0.03, respectively). In contrast, prenatal betamethasone exposure had no effect on basal GFR, Na+ excretion, or the percentage of the Na+ load excreted during the experiment in females. Systemic infusions of ANG-(1-7) at 9 ng.min(-1).kg(-1) for 2 h had minimal effects on basal GFR, renal plasma flow, and Na+ excretion in males but increased Na+ excretion in females. However, the percentage of Na+ load excreted during ANG-(1-7) infusion did not change in prenatal betamethasone-exposed females (113.1 +/- 14.2 vs. 98.1 +/- 12.2%) compared with the significant increase in vehicle females (139.2 +/- 22.3 vs. 92.2 +/- 7.5%) (P = 0.01). The data indicate that antenatal betamethasone exposure produces gender-specific alternations in renal function and thus suggest that different mechanisms underlie the antenatal steroid-induced elevations in blood pressure in male and female offspring.

Cortisol infusion in late-gestation hypothalamo-pituitary disconnected sheep fetus restores pituitary cell responsiveness to arginine vasopressin.

Corticotrophs in the fetal sheep become increasingly responsive to arginine vasopressin (AVP) in late gestation. We previously reported that this may be due in part to corresponding increases in signal transduction (inositol 1,4,5-trisphosphate, IP(3)). These ontogenic changes are prevented by hypothalamo-pituitary disconnection (HPD), which also prevents fetal plasma cortisol concentrations from increasing in late gestation. This led us to hypothesize that cortisol is involved in mediating the changes in pituitary responsiveness. HPD was performed on fetal sheep at 120 days gestational age (dGA). Half of the HPD fetuses were infused with cortisol for 3 days beginning at 135-137 dGA (HPD+C). The remaining HPD fetuses and a group of sham-operated control fetuses were infused with saline. Pituitary cells were isolated and cultured. After 48 h, a subset of cells was stimulated with 100 nM AVP for 2 h, and the medium was collected for ACTH analysis. Another subset of cells was stimulated with 100 nM AVP for 30 min, and the formation of IP(3) was determined. Plasma cortisol concentrations increased rapidly within the first 6 h after infusion (5.2 +/- 1.9 to 29.7 +/- 4.9 ng/ml) but did not increase thereafter. Cells from HPD+C and sham-operated fetuses secreted significantly more ACTH than those from HPD fetuses (% increase from control: 33.0 +/- 8.8%, 47.9 +/- 10.6%, and 11.9 +/- 2.4%, respectively). IP(3) formation was significantly increased in cells from HPD+C and sham-operated compared with HPD fetuses (% increase from control: 17.7 +/- 4.4%, 18.9 +/- 4.3%, and 4.6 +/- 1.5%, respectively). These findings support the idea that cortisol plays a role in mediating the increase in pituitary responsiveness to AVP in the late-gestation fetal sheep.

Plasma and renal renin concentrations in adult sheep after prenatal betamethasone exposure.

This study examined whether renin expression and secretion and plasma angiotensin II (Ang II) levels were altered in adult sheep exposed to antenatal betamethasone. Pregnant sheep received injections of 0.17 mg/kg betamethasone or vehicle, at 80 and 81 days of gestation, and offspring were studied at 6 and 18 months of age. At 6 months, plasma prorenin concentrations were significantly lower in betamethasone animals (4.63 +/- 0.64 vs 7.09 +/- 0.83 ng angiotensin I/mL/h, P < .01). The percentage of plasma active renin was significantly higher in the betamethasone group (31.93 +/- 4.09% vs 18.57 +/- 2.79%, P < .01). Plasma and renocortical renin levels were similar in both groups at 18 months, but plasma renin activity was lower than at 6 months. Ang II levels were suppressed by betamethasone. The data indicate that prenatal exposure to betamethasone alters processing and secretion of renin in offspring at 6 months, but that this difference is not apparent at 18 months.

Thyroid hormone regulates renocortical COX-2 and PGE2 expression in the late gestation fetal sheep.

Cyclooxygenase 2 (COX-2) is important for development of the fetal kidney. Precisely how renal COX-2 expression is regulated in fetal life is unclear. The hypothesis that thyroid hormone positively regulates COX-2 and PGE(2) levels in the late gestation fetal kidney cortex was tested. Sham, thyroidectomized (TX), and TX + thyroid hormone replacement (R) fetal sheep were studied. TX was performed at 120 days gestational age (dGA). TX + R fetuses were continuously infused with thyroxine from 3 days after surgery until study completion. Fetal kidney cortex was obtained at 137 dGA for measurement of renal cyclooxygenase type-2 (COX-2) protein and PGE(2) metabolites. Renocortical COX-2 and PGE(2) levels were significantly lower in TX compared with sham and TX + R fetuses. There were no differences between sham and TX + R fetuses. These findings demonstrate that thyroid hormone positively regulates renal COX-2 and PGE(2) expression in the late gestation fetal sheep kidney.

Thyroid hormone replacement normalizes renal renin and angiotensin receptor expression in thyroidectomized fetal sheep.

Previous studies have suggested that thyroid hormone influences maturation of the renin-angiotensin system (RAS) and cardiovascular function in the late-gestation fetal sheep. To further examine the importance of thyroid hormone in this regard, we used the technique of thyroidectomy (TX) to remove endogenous thyroid hormone from the circulation and then replaced it with physiological amounts of exogenous thyroxine. We hypothesized that the previously observed changes in RAS activity and cardiovascular function associated with TX would be normalized. TX was performed at 120 days of gestational age (dGA), and control fetuses were sham operated. After 3 days of recovery, TX fetuses were continuously intravenously infused with thyroxine until delivery by cesarean section close to term (around 138 dGA). Immediately before necropsy, fetuses were infused with isoproterenol, and the hemodynamic responses were noted. Thyroid hormone replacement normalized not only plasma triiodothyronine (T3) and thyroxine (T4) levels but also the TX-induced decreases in renal renin mRNA and renal renin content. Renal ANG II subtype receptor expression levels were also normalized for both mRNA and protein. Decreased basal heat rate and systolic blood pressure associated with TX returned to normal following replacement; however, changes in mean blood pressure and isoproterenol-induced changes in mean blood pressure were not altered. These findings demonstrate that replacement of thyroid hormone in hypothyroid sheep fetuses can restore renal ANG II receptor and renin expression and secretion to normal.

Ontogeny and effects of hypothalamic pituitary disconnection on formation of inositol trisphosphate in fetal sheep pituitary cells.

In late gestation fetal sheep, the pituitary becomes increasingly responsive to stimulation by arginine vasopressin (AVP). This change appears to be one important factor mediating the plasma cortisol surge, a critical developmental event. It is not known precisely why pituitary corticotropes become more responsive at this time. In this study we examined the possibility that changes in second messenger generation [inositol trisphosphate (IP(3))] are responsible. Two studies were undertaken. The first was an ontogeny study, where pituitaries were isolated from 100-, 120-, and 140-d gestational age (dGA) fetal sheep. Cells were cultured, stimulated with AVP, and the formation of IP(3) assessed. The amount of IP(3) generated increased with gestational age (percent increases from unstimulated controls were 4.6, 11.5, and 21.5 for 100, 120, and 140 dGA, respectively), with significant differences between the 140-dGA group and both earlier groups apparent. The second study examined the impact of 120-dGA hypothalamo-pituitary disconnection (HPD), which prevents corticotrope maturation, on responsiveness of pituitary cells isolated from 140-dGA fetuses. Cells were stimulated with AVP, and the formation of IP(3) and secretion of ACTH were assessed. Significantly less IP(3) was formed, and ACTH secreted in cells from HPD compared with control fetuses (IP(3) and ACTH levels were 50% and 35% lower, respectively). Results from the HPD study demonstrate that the ontogenic changes in IP(3) after AVP require an intact hypothalamic-pituitary-adrenal axis. These findings suggest that heightened second messenger generation may be a key reason for increased ACTH secretory responsiveness to AVP in the late gestation sheep fetus.

Combined thyroidectomy and renal denervation suppress renin expression and secretion in fetal sheep.

BACKGROUND AND OBJECTIVES: Activity of the fetal renin-angiotensin system (RAS) is developmentally regulated, increasing in late gestation toward term. Thyroid hormone and the renal nerves are both important modulators of renal RAS maturation; however, ablation of either influence alone does not totally block the aforementioned developmental late gestation increase in RAS in fetal sheep. In the current study, we used the technique of thyroidectomy combined with bilateral renal denervation (TX+D), which removes thyroid hormone from the circulation and abolishes effects of renal nerve activity, to determine if simultaneous removal of their effects on the kidney would markedly alter renin expression and secretion in late gestation. METHODS: TX+D was performed at 120 days of gestation age (dGA). Control fetuses were sham-operated. Immediately before necropsy (approximately 138 dGA), fetuses were infused with isoproterenol to examine plasma active and prorenin changes in response to beta-adrenergic stimulation. RESULTS: TX+D decreased plasma thyroid hormone concentrations, renal renin mRNA, renal active and prorenin levels, and plasma active and prorenin concentrations. Isoproterenol-induced increases in plasma active renin were also reduced in TX+D fetuses. TX+D did not alter renal angiotensin (Ang) II subtype receptor (AT2) expression close to term. CONCLUSION: These findings suggest that TX+D synergize in the suppression of fetal renin expression.

Infusion of ACTH stimulates expression of adrenal ACTH receptor and steroidogenic acute regulatory protein mRNA in fetal sheep.

The late-gestation plasma cortisol surge in the sheep fetus is critical for stimulating organ development and parturition. Increased adrenal responsiveness is one of the key reasons for the surge; however, the underlying mechanisms are not fully understood. Our recent studies suggest that ACTH-mediated increased expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) may play a role in enhancing responsiveness. Hence, we examined effects of ACTH infusion in fetal sheep on mRNA expression of these two mediators of adrenal responsiveness and assessed the functional consequences of this treatment in vitro. Fetuses of approximately 118 and 138 days of gestational age (dGA) were infused with ACTH-(1-24) for 24 h. Controls received saline infusion. Arterial blood was sampled throughout the infusion. Adrenals were isolated and analyzed for ACTH-R and StAR mRNA, or cells were cultured for 48 h. Cells were stimulated with ACTH, and medium was collected for cortisol measurement. Fetal plasma ACTH and cortisol concentrations increased over the infusion period in both groups. ACTH-R mRNA levels were significantly higher in ACTH-infused fetuses in both the 118 and 138 dGA groups. StAR mRNA increased significantly in both the 118 and 138 dGA groups. Adrenal cells from ACTH-infused fetuses were significantly more responsive to ACTH stimulation in terms of cortisol secretion than those from saline-infused controls. These findings demonstrate that increases in circulating ACTH levels promote increased expression of ACTH-R and StAR mRNA and are coupled to heightened adrenal responsiveness.

Ontogeny and effects of thyroid hormone on beta1-adrenergic receptor mRNA expression in ovine fetal kidney cortex.

OBJECTIVE: Previous studies indicate that thyroidectomy (TX) decreases renin gene expression in ovine fetal renal cortex in late gestation. Fetal ovine renin-containing renocortical cells become increasingly responsive to beta-adrenergic stimulation as gestation proceeds. Increases in plasma thyroid hormone concentrations parallel this change, suggesting that there is a positive developmental relationship between the two. To examine this hypothesis, we determined the ontogeny of beta1-adrenergic receptor (beta1R) mRNA expression, and the effect of thyroid hormone on in vivo and in vitro expression in fetal sheep. METHODS: Renocortical tissue was obtained from naive, TX, and sham-operated fetuses to determine beta1R mRNA levels. Renin-containing renocortical cells from TX or sham fetuses were treated with isoproterenol (Iso) or forskolin (FSK) for analysis of cellular cyclic adenosine monophosphate (cAMP) levels. Renocortical cells from naive fetuses were treated with triiodothyronine (T3) to assess cellular beta1R mRNA levels. Fetal plasma thyroxine (T4) level was determined. RESULTS: Renocortical beta1R mRNA expression increased significantly between 100 and 140 days' gestational age (dGA), while TX attenuated this increase (P <.01). Renocortical cellular cAMP levels were higher in sham compared to TX fetuses following incubation with Iso or FSK (P <.05). Cells incubated with T3 exhibited significantly increased beta1R mRNA expression (P <.05). CONCLUSION: The data suggest that thyroid hormone may be involved in modulating ovine fetal renocortical beta1R gene expression during development. We speculate that the increased beta1R mRNA expression in renal cortical cells as development progresses may mediate the increases in renin gene response to beta-adrenergic stimulation in late gestation.

Developmental changes in adrenocorticotrophin (ACTH)-induced expression of ACTH receptor and steroid acute regulatory protein mRNA in ovine fetal adrenal cells.

OBJECTIVES: Adrenocorticotrophin (ACTH) plays an important role in mediating the increase in cortisol output in the late gestation sheep fetus. At the adrenal itself, heightened expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) appear to be important parallel changes. This study examined how ACTH affects ACTH-R and StAR mRNA expression, and cortisol production in adrenocortical cells isolated from fetuses of varying gestational age (dGA). We hypothesized that the ability of ACTH to stimulate its receptor and StAR mRNA expression would be greater close to term than earlier in development. METHODS: Adrenals were obtained from fetuses (100-105, 120, or 135-139 dGA), and the cortical cells were dispersed. After 3 days of culture, cells were stimulated with ACTH(1-24), and the cells and medium were collected at different time points (0, 3, 6, 9, 12, and 24 hours) for measurement of cortisol and ACTH-R and StAR mRNA. RESULTS: Cortisol secretion was increased after ACTH treatment in all three age cohorts. Cells from the 135-139 dGA group secreted the most cortisol, followed by the 100-105 and then the 120 dGA groups (P <.05). ACTH-R mRNA levels before and after ACTH were higher in the late compared to both earlier groups. StAR mRNA levels before and after ACTH were higher in the 100-105 and 135 than in the 120 dGA group. The time to peak ACTH-R mRNA response was age-dependent, with the 100-105 dGA cells taking longer to attain maximum levels. Maximal StAR mRNA levels were not age-related. CONCLUSION: The data suggest that ACTH-R and StAR are indeed key mediators of fetal adrenocortical responsiveness, and that ACTH is able to up-regulate responsiveness, and hence cortisol production, by increasing their expression.

Thyroid hormone modulates renin and ANG II receptor expression in fetal sheep.

Fetal renin-angiotensin system (RAS) activity is developmentally regulated, increasing in late gestation toward term. At the same time, fetal hemodynamic parameters change, with blood pressure increasing and heart rate decreasing. During this period, fetal plasma thyroid hormone concentrations also increase significantly. In this study we utilized the technique of thyroidectomy (TX), which removes thyroid hormone from the circulation, to investigate the importance of thyroid hormone on the developmental changes in the RAS (in plasma, kidney, heart, and lung) and hemodynamic regulation in fetal sheep. TX was performed at 120 days of gestational age (dGA), and control fetuses were sham operated. Immediately before necropsy ( approximately 137 dGA), fetuses were infused with isoproterenol and the hemodynamic responses were noted. TX significantly decreased plasma thyroid hormone concentrations and renal renin mRNA and renal active renin levels but did not change fetal plasma active renin levels. TX decreased both angiotensin II receptor subtype 1 (AT1) mRNA and protein levels in kidney and lung but not in the left ventricle. TX also was associated with increased ANG II receptor subtype 2 (AT2) mRNA and protein at the 44-kDa band in kidney, whereas AT2 protein was decreased at the 78-kDa level in kidney and lung tissue only. TX fetuses had significantly lower basal mean arterial blood pressures (MAP) and heart rates than controls. Isoproterenol infusion decreased MAP in TX fetuses. These findings support the hypothesis that thyroid hormone is important in modulating maturation of RAS and cardiovascular function in the late-gestation fetal sheep.

Hypothalamic-pituitary disconnection in fetal sheep blocks the peripartum increases in adrenal responsiveness and adrenal ACTH receptor expression.

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At approximately 138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 +/- 1.2 vs. 47.3 +/- 11.1 ng.ml(-1).2 h(-1)) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.